This project is aimed towards understanding the mechanisms which mediate and regulate Ca-2+ signaling in salivary gland cells. Neurotransmitter stimulation of fluid secretion in salivary glands is mediated via a biphasic elevation in cytosolic [Ca-2+]; an initial transient increase due to internal release and a latter sustained increase due to Ca-2+ influx. Sustained fluid secretion is directly dependent upon the sustained elevation of [Ca-2+] and thus on Ca-2+ influx. Recently, our efforts have been focused on the Ca-2+ influx mechanism in salivary gland cells, which appears to be a mediated via store-operated Ca-2+ entry (SOCE) that is ubiquitously present in many other non-excitable cells. The molecular mechanism(s) of this influx has not yet been determined in any cell type. Recently, the transient receptor potential (TRPC) family of ion channel proteins have been proposed as molecular components of the store-operated Ca-2+ influx channel (SOCC). However, the physiological function(s) of the presently identified TRPCs has not yet been fully established. By expressing TRPC1 in vivo in rat SMG by using an adenovirus encoding hTrp1 (AdHA-hTrp1) and in salivary gland cell lines, we had previously reported that TRPC1 is involved in the regulation of store-operated calcium influx in salivary gland cells. In the past year our major effort has continued to be towards characterizing SOCE and identifying the role of TRPC1 in the SOCE mechanism of salivary gland cells. Consistent with our previous studies, we have now reported that TRPC1 is an integral component of SOCC. More importantly, our studies demonstrate that it is involved in the Ca-2+ dependent feedback inhibition of SOCC. We have shown that CaM mediates this feedback inhibition by binding to a domain, aa758-793, in the C-terminus of TrpC1. This study demonstrated for the first time a possible mechanism for the Ca-2+ dependent feedback inhibition of SOCE. Although TRPC1 appears to be required for store-operated calcium channel (SOCC) function, its exact role in SOCC is not known. Based on the available data it is possible that TRPC1 could function either as a regulator of SOCC or as a pore-forming subunit. Towards resolving this we have used a mutagenesis approach to demonstrate that TRPC1 directly contributes to SOCC activity and that acidic amino acid residues in the TRPC1 putative pore domain (between the 5th and 6th TM regions) are involved in SOCC function. Ongoing studies in the lab aim to identify the specific amino acid residue(s) in TRPC1 that determine the calcium permeability of SOCC. These data demonstrate for the first time that a TRPC protein is a pore-forming subunit of SOCC. Thus, these studies represent a major advancement in our understanding of the mechanism of SOCE in salivary gland cells. SOCC has been proposed to consist of TRPC dimers or multimers. In this reporting period we have shown that epitope-tagged TRPC1 proteins co-imunoprecipitate. Further, by using yeast-two hybrid screen and GST-pull down assays, we have determined that TRPC1 monomers interact via their N-terminus, likely at the first ankyrin-repeat domain. Importantly, expression of the TRPC1 N-terminus exerts a dominant negative effect on SOCC activity and disrupts the interaction of TRPC monomers. Thus, we propose that multimerization of TRPC1 monomers is required for SOCC function and that this is achieved via interaction of the first N-terminal ankyrin repeats. In the coming year we will examine heteromeric interaction of TRPC1 with other TRPC proteins. We have reported earlier that TRPC1 and 3 are assembled in multimolecular signaling complexes associated with caveolin-1. We have now identified the binding site for caveolin-1 in the TRPC1 N-terminus and shown that caveolin-1 is required for the plasma membrane localization of TRPC1 and TRPC3. Further, yeast-two hybrid analysis demonstrated that TRPC1 interacts with the SNARE proteins VAMP2 and SNAP. Further studies showed that VAMP2 is involved in the trafficking of TRPC3 and caveolin to the plasma membrane. Disruption of VAMP2 with tetanus toxin altered the plasma membrane localization of TRPC3 and caveolin. We propose that TRPC1 and 3 are delivered to the plasma membrane via an exocytotic pathway. Currently, we are examining whether the assembly into caveolar signaling complexes occurs at the plasma membrane or intracellularly. We will also determine whether retention of TRPC1 signaling complexes in the plasma membrane requires caveolin-1. We propose that the regulation of SOCE occurs via protein-protein interactions in the TRP-associated signaling complex. Thus, it is essential to identify its protein components. Towards this, we have initiated two approaches: a yeast-two hybrid screen and a proteomics-based screen. In the latter we have immunopurified TRPC3 and are examining the proteins that are associated with it by using 2D-MS-MS analysis. Both methods have yielded novel as well as known proteins. In the coming year we will continue these studies and also confirm the initial observations with more detailed experiments.